The type 3 serotonin (5-HT3) receptor is a ligand-gated ion channel. Pharmacological studies had shown that the 5-HT3-receptor facilitates dopamine (DA) release in the nucleus accumbens (NAc). The 5-HT3-receptor antagonists reduce extracellular DA induced by 5-HT3-receptor agonists, drugs of abuse and by the direct stimulation of dopaminergic neurons. The source of the 5-HT3-receptor that regulates DA release is unknown. Although pharmacological studies suggest that the 5-HT3-receptor may be present in the NAc and ventral tegmental area (VTA), prior anatomical studies have revealed few or no 5-HT3-receptor binding sites in these regions. By using in situ hybridization histochemistry, we detected expression of the functional 5HT3A subunit, but not 5HT3B subunit, in the rat midbrain. The 5HT3A were found in the VTA, parabrachial pigmented nucleus, substantia nigra pars compacta (SNC), substantia nigra pars lateralis (SNL), prerubral field, medial and supra mammillary nucleus, and interpeduncular nucleus (IP). To determine the cellular phenotype of 5HT3A expressing neurons, we used double-labeling techniques and determined that dopaminergic cells express the 5HT3A subunit in the SNC and VTA. In addition, the 5HT3A subunit was found to be expressed in GABAergic neurons located in the substantia nigra pars reticulata, SNL and VTA. The localization of 5HT3A subunit transcripts in dopaminergic neurons suggests that serotonin, through 5-HT3-receptors present in the midbrain, may regulate the release of DA. In addition, expression of the 5HT3A subunit in midbrain GABAergic neurons suggests that serotonin might modulate dopaminergic neuronal activity via 5-HT3-receptors distributed in local GABAergic neurons.